© 2017. Published by The Company of Biologists Ltd.

CCR2 3′UTR functions as a competing endogenous RNA to inhibit breast cancer

metastasis

Jinhang Hu 1,2, Xiaoman Li 3, Xinwei Guo1,2, Qianqian Guo 1,2, Chenxi Xiang 1,2,

Zhiting Zhang 1,2, Yingying Xing 1,2, Tao Xi 1,2,*, Lufeng Zheng 1,2,*

1 School of Life Science and Technology, China Pharmaceutical University, Nanjing,

210009, People’s Republic of China

2 Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical

University, Nanjing, 210009, People’s Republic of China

3 Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia

Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023,

People’s Republic of China

* Correspondence to: Tao Xi, Tel: + 86 25 8327 1022; Fax: +86 25 8327 1022; E-mail:

xitao18@hotmail.com. Lufeng Zheng: Email: zhlf@cpu.edu.cn.

Keywords: CCR2, 3′UTR, ceRNA, metastasis, breast cancer

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JCS Advance Online Article. Posted on 17 August 2017

Abstract

Diverse RNA transcripts acting as competing endogenous RNAs (ceRNAs) can co-regulate

each other’s expression by competing for shared microRNAs. CCR2 protein, the receptor of

CCL2, is implicated in cancer progression. However, our results showed that higher CCR2

mRNA level was remarkably associated with prolonged survival of breast cancer patients.

The conflicting results prompt us to study the non-coding function of CCR2 mRNA. We

indicated that CCR2 3′UTR inhibited MDA-MB-231 and MCF-7 cells metastasis by

repressing EMT in vitro and suppressed breast cancer metastasis in vivo. Mechanistically,

CCR2 3′UTR modulated RhoGAP protein STARD13 expression via acting as a STARD13

ceRNA in a microRNA-dependent and protein coding-independent manner. CCR2 3′UTR

blocked the activation of RhoA-ROCK1 pathway which is the downstream effector of

STARD13 and thus decreased the phosphorylation level of MLC and formation of F-actin.

Additionally, the function of CCR2 3′UTR was dependent on STARD13 expression. In

conclusion, our results confirmed that CCR2 3′UTR acts as a metastasis suppressor by acting

as a ceRNA for STARD13 and thus inhibiting RhoA → ROCK1 → MLC → F-actin pathway

in breast cancer cells.

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1. Introduction

Breast cancer is the most common malignancy among the women worldwide (Sui et al.,

2015). Metastasis is a complex process leading to dissemination of cancer cells from the

primary tumor to distant organ (Gilkes et al., 2014) and it results in the vast majority of

deaths of patients with breast cancer (Papageorgis et al., 2015). Thus, it is an urgent need to

elucidating the molecular mechanism contributing to breast cancer metastasis.

CCL2 is a member of the CC chemokine family and acts on its target cells by binding to

the cognate cysteine-cysteine chemokine receptor 2 (CCR2), a member of the

seven-transmembrane, G protein-coupled receptor family which is the main signaling partner

of CCL2 (Lee et al., 2009). Recently, the CCL2/CCR2 axis has attracted increasing interest

due to its association with the tumor progression (Lim et al., 2016). On one side, CCL2 can

be synthesized by metastatic tumour cells and stromal cells in the tumor microenvironment,

which is critical for recruiting a sub-population of CCR2-expressing monocytes or

macrophages that enhance the subsequent extravasation of tumour cells (Qian et al., 2011).

On the other hand, CCR2 is expressed in various cancer types and regulates CCL2-induced

breast cancer cell survival and motility through MAPK- and Smad3-dependent mechanisms

(Fang et al., 2012). In addition, CCL2/CCR2 could induce STAT3 activation and

epithelial-mesenchymal transition (EMT) of PCa cells (Izumi et al., 2013), CCL2/CCR2 axis

could promote metastasis of nasopharyngeal carcinoma by activating ERK1/2-MMP2/9

pathway (Yang et al., 2016). These results support that the CCR2 protein, as a receptor,

together with its cognate ligand CCL2 promotes cancer metastasis. However, Kaplan-Meier

analysis of disease-specific survival for breast cancer patients stratified by CCR2 mRNA

expression showed that higher expression of CCR2 mRNA was significantly correlated with

longer survival. Therefore, we assumed that CCR2 transcript may hold distinct functions with

its protein in breast cancer progression.

The competing endogenous RNAs (ceRNAs) hypothesis posits that all RNA transcripts

can modulate each other’s expression by competing for shared microRNAs, thus acting as

ceRNAs (Cesana et al., 2011; Karreth et al., 2011; Salmena et al., 2011; Sumazin et al., 2011;

Tay et al., 2011; Tay et al., 2014). Multiple non-coding RNA species, including small

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non-coding RNAs, pseudogenes (Zheng et al., 2015), lncRNAs (Cesana et al., 2011; Chou et

al., 2016; Liu et al., 2013) and circular RNAs (circ RNAs) can possess ceRNA activity.

mRNA transcripts may act as tumor suppressors or oncogenes through their ceRNA activity,

this activity may be complementary or even distinct from their protein-coding function (Tay

et al., 2014). For example, the ZEB2 protein, promotes the progression of glioma (Li et al.,

2016a) and lung cancer (Jiao et al., 2016); however, as a PTEN ceRNA, the ZEB2 mRNA

displays tumor-suppressive properties in melanoma cells (Karreth et al., 2011). To give the

potentially distinct function of the CCR2 transcript, we examined whether CCR2 mRNA

exerts a tumor-suppressive function in breast cancer through its ceRNA activity.

STARD13 (also called DLC2), a unique RhoGAP (RhoGTPase-activating protein), is

underexpressed in some types of cancer and suppresses cytoskeleton reorganization, cell

migration and transformation by inhibiting RhoA activity through its RhoGAP domain

(Ching et al., 2003; Leung et al., 2005; Lin et al., 2010). Active (GTP-loaded) RhoA binds to

and activates ROCK1 (Rho-associated coiled-coil-forming kinase 1). RhoA-bound ROCK1

phosphorylates and activates MLC (myosin light chain), increasing the level of pMLCS19

which is required to coordinate the formation of stress fibers (Gilkes et al., 2014). Increased

formation of the filamentous actin (F-actin) cytoskeleton and enhanced contractility are

critical to cell migration (Stricker et al., 2010). microRNAs that target CCR2 mRNA have not

been reported before. Here, TargetScan (http://www.targetscan.org/) was used to predict the

microRNA binding sites in CCR2 3′-untranslated region (3′UTR), and three microRNA-125b

binding sites were found to exist in CCR2 3′UTR. Our laboratory has proved that

microRNA-125b can promote metastasis of MCF-7 and MDA-MB-231 cells by targeting

STARD13 (Tang et al., 2012). Furthermore, our recent study described a

STARD13-correlated ceRNA network which possesses a metastasis-suppressive role in breast

cancer (Li et al., 2016b). Thus, we deduced that CCR2 mRNA acts as a tumor suppressor in

breast cancer through its ceRNA activity to promote STARD13 expression in a

protein-independent but microRNA-dependent way.

Our study revealed that CCR2 3′UTR inhibited the EMT and modulated STARD13

expression in MDA-MB-231 and MCF-7 cells. CCR2 3′UTR blocked RhoA-ROCK1

signaling which is the downstream effector of STARD13 and thus decreased the

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phosphorylation of MLC and the formation of F-actin. Taken together, these results delineate

a molecular mechanism by which CCR2 3′UTR regulates STARD13 expression and further

inactivates RhoA-ROCK1 pathway which is required for breast cancer metastasis.

2. Results

2.1 CCR2 3′UTR displays tumor-suppressive properties in breast cancer cells

We first investigated the roles of CCR2 3′UTR in breast cancer development. Firstly,

Kaplan-Meier survival analysis which was performed using KM-plotter (Gyorffy et al., 2010)

revealed that CCR2 mRNA level was positively correlated with the longer overall survival

(OS) (p= 0.0011) (Fig. 1A), distant metastasis free survival (DMFS) (p= 0.018) (Fig. 1B) and

relapse free survival (RFS) (p= 2.9e-15) (Fig. 1C) of breast cancer patients. The results seem

to contradict the idea that CCR2 enhances CCL2-induced breast cancer cells survival and

motility, which promoted us to explore whether CCR2 mRNA holds distinct function from its

protein-coding function. Cells migration was measured in MCF-7 and MDA-MB-231 cells

with CCR2 3′UTR or empty vector transfection by wound healing and transwell migration

assays. Overexpression of CCR2 3′UTR remarkably decreased the migration of MCF-7 and

MDA-MB-231 cells compared with control vector (Fig. 1D and E). Adhesion of tumor cells

to extra-cellular and basement membranes plays a critical role in the initial step in the

invasive process (Tang et al., 2012). The adhesion ability of CCR2 3′UTR-transfected cells

was decreased in MCF-7 and MDA-MB-231 cells (Fig. 1F). A similar trend was observed in

transwell migration assay (Fig. 1G and H). Additionally, matrigel invasion chambers were

used to examine the role of CCR2 3′UTR towards tumor invasiveness. The invasive ability of

CCR2 3′UTR-transfected cells was markedly decreased compared to cells treated with

control vector (Fig. 1G and I). In addition, cell cycle analysis showed that no relevant

changes were detected upon CCR2 3'UTR ectopic expression in the percentage of cells in

G0/ G1, G2/ M and S phases (Fig. S1A-D). Further cell apoptosis analysis indicated that

CCR2 3′UTR overexpression had no significant effect on cell apoptosis (Fig. S1E-H). These

results suggest that ectopic expression of CCR2 3′UTR suppresses breast cancer cells

metastasis and has no effect on cell cycle progression and apoptosis.

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2.2 Ectopic expression of CCR2 3′UTR inhibits EMT process

Epithelial-mesenchymal transition (EMT) is the most critical step in tumor metastasis

(Biddle and Mackenzie, 2012; Kong et al., 2008). We further sought to determine whether

CCR2 3′UTR could reverse EMT phenotype. As shown in Fig. 2A and 2B, the ectopic

expression of CCR2 3′UTR or STARD13 3′UTR resulted in up-regulation of epithelial

marker E-cadherin and down-regulation of mesenchymal markers including N-cadherin,

α-SMA in MCF-7 cells. Furthermore, TGF-β1-induced EMT was used in epithelial MCF-7

cells. 48 h after TGF-β1 treatment, MCF-7 cells exhibited lower levels of E-cadherin and

higher N-cadherin, which was reversed by CCR2 3′UTR overexpression (Fig. 2C).

Additionally, after TGF-β1 treatment, MCF-7 cells underwent a dramatic morphological

change, from cobblestone-like epithelial structure to fibroblastoid spindle-shaped cells. This

morphological transition is accompanied by E-cadherin downregulation. Furthermore,

transfection with CCR2 3′UTR significantly reduced the acquisition of mesenchymal

characteristics during the TGF-β1-induced EMT of MCF-7 cells (Fig. 2D). The ectopic

expression of CCR2 3′UTR or STARD13 3′UTR downregulated mesenchymal markers

expression including N-cadherin, Vimentin in MDA-MB-231 cells, whereas α-SMA

expression was not affected in MDA-MB-231 cells (Fig. 3A and B). Identical results were

obtained by immunofluorescence staining analysis (Fig. 3C and D). Taken together, these

results demonstrate that CCR2 3′UTR could block the TGF-β1-induced EMT of MCF-7 cells

and induce MET in MCF-7 and MDA-MB-231 cells.

2.3 CCR2 3′UTR modulates expression of STARD13

Based on the fact that a series of common MREs (miRNA response elements) exist in

the 3′UTR of CCR2 and STARD13, we assumed that CCR2 is a ceRNA candidate of

STARD13 which has proven to be a tumor suppressor. We first examined whether CCR2

3′UTR could modulate STARD13 expression. Ectopic expression of CCR2 3′UTR did

significantly upregulate the mRNA levels of CCR2 and STARD13 in MDA-MB-231 cells

(Fig. 4A) and MCF-7 cells (Fig. S2A). Similarly, STARD13 3′UTR could also upregulate the

mRNA levels of CCR2 and STARD13 (Fig. 4B and Fig. S2B). Moreover, STARD13 and

CCR2 protein levels were increased in CCR2 3′UTR-transfected (Fig. 4C and Fig. S2C) and

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STARD13 3′UTR-transfected (Fig. 3D and Fig. S2D) breast cancer cells. In contrast,

knockdown of CCR2 reduced STARD13 mRNA levels and knockdown of STARD13 led to

downregulation of CCR2 mRNA levels in MDA-MB-231 cells (Fig. 4E) and MCF-7 cells

(Fig. S2E). Importantly, STARD13 and CCR2 protein levels were reduced following

knockdown of CCR2 or knockdown of STARD13 in MDA-MB-231 cells (Fig. 4F) and

MCF-7 cells (Fig. S2F). These results indicate that CCR2 and STARD13 can regulate each

other’s expression.

2.4 CCR2 is a potential target of microRNA-125b

To elucidate which microRNA mediates the CCR2-STARD13 crosstalk, we interrogated

the common MREs in the 3′UTRs of STARD13 and CCR2. TargetScan 6.2 predicted that

CCR2 and STARD13 shared four microRNA families and contained a total of 6 and 9 MREs

for these microRNAs respectively, including microRNA-125b (Fig. 5A). Our previous study

has indicated that microRNA-125b induced breast cancer metastasis by targeting STARD13

directly (Li et al., 2016b), whereas the miR-125b/CCR2 axis should be validated. qRT-PCR

analysis confirmed the transfection efficiency of miR-125b mimics and miR-125b inhibitor in

MDA-MB-231 (Fig. 5B and C) and MCF-7 cells (Fig. S3A and S3B). TargetScan 6.2

analysis indicated that STARD13 contains four miR-125b binding site on its 3′UTR and two

of these four sites were predicted to be 8mer site (Fig. 5D). To validate whether CCR2 is

regulated by miR-125b, TargetScan 6.2 was adopted to predict targets of miR-125b and the

3′UTR of CCR2 was predicted to contain three complementary sites for the seed region of

miR-125b. One of these three sites, starting at nt 169 of the CCR2 3′UTR, was predicted to

be 8mer site. CCR2 3′UTR fragments containing wild-type (CCR2-WT) of this miR-125b

target site or mutant binding site (CCR2-MUT) was introduced downstream of the luciferase

reporter gene in pMIR-reporter vector (Fig. 5D). CCR2-WT or CCR2-MUT was

co-transfected with miR-125b mimics in MDA-MB-231 and MCF-7 cells (Fig. 5E and Fig.

S3C), the relative luciferase activity of CCR2-WT was attenuated, while the activity of

CCR2-MUT was unaffected. To validate the direct binding between miR-125b and CCR2

3′UTR or STARD13 3′UTR, we performed an RNA immunoprecipitation (RIP) assay using

antibody against Ago2, which is the core component of the RNA-induced silencing complex,

to pull down microRNAs associated with CCR2 3′UTR or STARD13 3′UTR. qRT-PCR

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analysis demonstrated that the CCR2 3′UTR or STARD13 3′UTR RIP in MDA-MB-231(C)

cells (stably transfected with CCR2 3′UTR) and MDA-MB-231(S) cells (stably transfected

with STARD13 3′UTR) were significantly enriched for miR-125b compared to the

MDA-MB-231(V) (stably transfected with an empty vector) (Fig. 5F). CCR2 and STARD13

mRNA levels were significantly decreased or increased with miR-125b mimics or inhibitor

transfection respectively in MDA-MB-231 (Fig. 5G and H) and MCF-7 cells (Fig. S3D and

S3E). Furthermore, overexpression of miR-125b resulted in a significant reduction in protein

levels of CCR2 and STARD13 (Fig. 5I and Fig. S3F) and knockdown of miR-125b led to an

increase in protein levels (Fig. 5J and Fig. S3G). Taken together, these data indicate that

CCR2 is a potential target of miR-125b.

2.5 MicroRNA dependency of CCR2 -STARD13 interaction

To further confirm that CCR2 acts as a STARD13 ceRNA, we must confirm that the

CCR2-STARD13 interaction is indeed mediated by microRNAs. Thus, siRNA against

DICER, a microRNA biogenesis protein, was utilized. The efficient knockdown of siDICER

and the concomitant downregulation of miR-125b have been revealed by our earlier study (Li

et al., 2016b). siCCR2 failed to inhibit STARD13 mRNA and protein expressions when

co-transfected with DICER siRNA, while CCR2 mRNA level was decreased. Similarly,

STARD13 knockdown diminished STARD13 mRNA level but unaffected CCR2 mRNA and

protein levels in siDICER-treated cells compared with NC (Fig. 6A and B, Fig. S4A and

S4B). Moreover, in the presence of siDICER, CCR2 3′UTR-mediated or STARD13 3′UTR

–mediated increase of STARD13 and CCR2 protein expression was lost (Fig. 6C and D, Fig.

S4C and S4D). To confirm the pivotal roles of miRNAs in the regulation between CCR2 and

STARD13, we performed an RIP assay on Ago2. As shown in Fig. 6E, overexpression of

CCR2 3′UTR in MDA-MB-231 cells led to the increased enrichment of Ago 2 on CCR2

3′UTR but substantially decreased enrichment on STARD13 3′UTR and this result suggested

that CCR2 3′UTR could compete with STARD13 transcripts for the Ago2-based

miRNA-induced repression complex. Notably, ectopic expression of CCR2 3′UTR with

mutation of all three predicted miR-125b binding sites did significantly upregulate the protein

level of CCR2 but no significantly change in STARD13 protein level (Fig. 6F). Importantly,

wound healing assay showed that CCR2 3′UTR with mutation of all three predicted

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miR-125b binding sites caused no significant decrease in the migration of MDA-MB-231

cells (Fig. 6G). These data indicate that CCR2 3′UTR-mediated regulation of STARD13 is

indeed dependent on microRNA and the miR-125b target sites within CCR2 3′UTR,

especially, are necessary for CCR2 3′UTR to inhibit the metastasis of breast cancer cells.

2.6 CCR2 3′UTR and STARD13 3′UTR suppress RhoA activity, MLC phosphorylation

and formation of stress fibers

To study whether CCR2 3′UTR could regulate the downstream signals of STARD13,

immunofluorescence staining of filamentous actin (F-actin) with rhodamine-labeled

phalloidin was performed and revealed that stress fiber formation was markedly inhibited in

CCR2 3′UTR- and STARD13 3′UTR-transfected cells (Fig. 7A and Fig. S5A). Additionally,

the levels of pMLCS19 were decreased significantly in CCR2 3′UTR-transfected (Fig. 7B and

Fig. S5B) or STARD13 3′UTR-transfected (Fig. 7C and Fig. S5C) cells. To further

determine whether RhoA or ROCK1 was involved in CCR2 3′UTR- or STARD13

3′UTR-mediated inhibition of MLC phosphorylation, qRT-PCR and western blot analyses

were performed and revealed that CCR2 3′UTR and STARD13 3′UTR had no effect on

expression of RhoA and ROCK1 in MDA-MB-231 cells (Fig. 7D-7G) and in MCF-7 cells

(Fig. S5D-S5G). Furthermore, G-LISA RhoA activation assay revealed that CCR2 3′UTR

transfected cells exerted a lower basal level of RhoA activity (Fig. 7H). In summary, these

data indicate that CCR2 3′UTR and STARD13 3′UTR inhibit the formation of F-actin and

MLC phosphorylation through regulating RhoA activity rather than RhoA and ROCK1

expression in human breast cancer cells.

2.7 CCR2 3′UTR decreased formation of stress fibers and MLC phosphorylation

dependent on STARD13

We observed that CCR2 3′UTR could inhibit formation of F-actin and MLC

phosphorylation. To further ascertain whether these observed effects are dependent upon

STARD13 expression, immunofluorescence staining of F-actin manifested that siSTARD13

could reverse CCR2 3′UTR-mediated inhibition on stress fiber formation in cells (Fig. 7I and

Fig. S5H), suggesting that CCR2 3′UTR induces cell migration via STARD13. Furthermore,

blocking STARD13 expression with siSTARD13 abolished CCR2 3′UTR-induced decrease

of pMLCS19 in MDA-MB-231 (Fig. 7J) and MCF-7 cells (Fig. S5I). Taken together, these

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results demonstrate that the decreased MLC phosphorylation and formation of stress fibers

induced by CCR2 3′UTR are STARD13 dependent.

2.8 CCR2 3′UTR inhibits breast cancer metastasis in a xenograft model

The antitumor function of CCR2 3′UTR in breast cancer was further confirmed in vivo.

qRT-PCR analysis demonstrated the efficient overexpression of CCR2 3′UTR in stably

transfected cells (Fig. 8A). MDA-MB-231(C) cells expressed more CCR2 and STARD13

protein (Fig. S6A) and fewer pMLC protein (Fig. S6B) than MDA-MB-231(V) cells. The

morphology were changed from angular and spindle to round in shape in MDA-MB-231 cells

stably transfected with CCR2 3′UTR (MDA-MB-231(C) cells) and the stress fiber formation

was inhibited in these cells (Fig. S6C). Additionally, stably transfected with CCR2 3′UTR

significantly decreased the cell migration in MDA-MB-231 cells (Fig. S6D). We

intravenously transplanted MDA-MB-231(V) or MDA-MB-231(C) into nude mice. As seen in

Fig. 8B, micro-computed to tomography (micro-CT) analysis revealed that CCR2 3′UTR

overexpression strikingly decreased the lung metastasis of MDA-MB-231 cells in vivo. The

carestream noninvasive optical imaging system was used for whole animal imaging, animals

injected with cells stably transfected with CCR2 3′UTR showed a reduced metastasis signals

in ventral views (Fig. 8C). Notably, CCR2 3′UTR suppressed pulmonary metastasis of

MDA-MB-231 cells, as shown by the bioluminescence of the excised tissues (Fig. 8D).

Again, histopathological analysis confirmed the inhibitory effect of CCR2 3′UTR on

pulmonary metastasis (Fig. 8E). Our in vivo data further strengthened the anti-metastatic

effects of CCR2 3′UTR in breast cancer.

3. Discussion

The present study shows that CCR2 3′UTR induces a microRNA-dependent increase of

STARD13 expression, leading to the downstream pathway inactivation that is manifested by

the decrease of RhoA activation, actin polymerization, MLC phosphorylation and thus

suppresses breast cancer metastasis (Fig. S7).

CCR2 is viewed as receptor of CCL2, and CCL2/CCR2 axis has been shown to play

crucial roles in cancer metastasis. Targeting this signaling pair is considered as a therapeutic

intervention. However, therapeutics obstructing this chemokine receptor pair displayed

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disappointing results in the clinic (Lim et al., 2016). Our analyses found that breast cancer

patients with higher CCR2 mRNA expression have a longer overall survival, distant

metastasis free survival and a longer relapse free survival. This suggests that high CCR2

mRNA is a marker for good prognosis in breast cancer. We put forward the inconsistency

between CCR2 transcript and CCR2 protein for the first time and demonstrated that CCR2

mRNA and protein exert different biological effects. The ceRNA hypothesis was used to

explain the molecular mechanisms of CCR2 mRNA. The ceRNA hypothesis provides a new

angle for studying the multilayered complexity of genes. Although it has been proposed that

ceRNA regulation is rare and can only exist in certain regimes of target and miRNA

abundance (Bosson et al., 2014; Denzler et al., 2014; Denzler et al., 2016), the discovery of

functional ceRNA regulation in diverse cancer progression by multiple independent groups

suggests that ceRNA may represent a widespread layer of gene regulation(Fang et al., 2013;

Kumar et al., 2014; Tay et al., 2014). In order to reveal the function of a gene, we should

integrate the classic “protein dimension” with an additional “ceRNA dimension” (Karreth et

al., 2011).

It has recently been reported that CCR2 knockdown in 4T1 and MCF-7 cells can block

CCL2-induced wound closure (Fang et al., 2012). In contrast, we proved that overexpression

of CCR2 3′UTR suppressed breast cancer cells migration and invasion. One possible

explanation for this discrepancy is that cells were not stimulated with CCL2 in our

experiments. In addition, CCR2 3′UTR had an influence on biomarkers of EMT, which

indicated that CCR2 3′UTR could affect breast cancer cells metastasis through suppressing

EMT process. Notably, a nude mice xenograft model verified the inhibitory role of CCR2

3′UTR on breast cancer metastasis in vivo.

Our previous studies have implicated STARD13 as a metastatic suppressor in MCF-7

and MDA-MB-231 cells in vitro and in vivo and miR-125b can negatively regulate the

expression of STARD13 by targeting STARD13 directly (Li et al., 2016b; Tang et al., 2012).

Although a series of experiments were performed to validate that CCR2 and STARD13 could

regulate each other’s expression, the ceRNA crosstalk is complex, various and multi-level.

We have to point out that this study did not concentrate some additional considerations for

ceRNA activity such as the abundance for key players, potential interplay with RBPs and

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RNA editing which may have effects on ceRNA crosstalk (Tay et al., 2014). Analyzing the

miRNA binding sites located in the CCR2 3′UTR has identified numerous miRNAs binding

with the CCR2 3′UTR. The change in the expression level of CCR2 can affect the level of

these potential CCR2-binding miRNAs, which in turn affect the level of other targets. In

addition to the direct interactions through shared miRNAs, secondary indirect interactions

may also have a profound effect on ceRNA regulation. The existence of distant ceRNAs was

recently introduced and it suggests that perturbation effects could propagate in the network

through a cascade of coregulated target RNAs and miRNAs that share targets, leading to

mutual effects between distant components(Nitzan et al., 2014), There is increasing evidence

confirming that ceRNAs crosstalk in large interconnected networks. Further studies are

needed to accurately indentify metastasis-related miRNA and mRNAs in the

CCR2-correlated ceRNA network.

TargetScan was employed as this algorithm considers MRE conservation between

mammals, TargetScan predicted that CCR2 and STARD13 are targets for four shared

microRNA families, among of them, number of miR-125b MRE in CCR2 3′UTR and

STARD13 3′UTR is more than other microRNAs, therefore miR-125b was used as a model to

explore the CCR2-STARD13 interaction. As expected, our results showed that miR-125b can

inhibited CCR2 expression. SiDICER was used to present an ideal system to evaluate

microRNA-dependent effects and the results showed that the CCR2-STARD13 is interaction

is a microRNA-mediated and MRE-dependent regulation. Especially, miR-125b target sites

within the CCR2 3′UTR are necessary to the CCR2-STARD13 interaction. Overexpression of

CCR2 3′UTR caused different multiple increase of CCR2 or STARD13 mRNA and their

protein levels and the possible explanation is that in contrast to transcription process, the

translation process is regulated by other mechanisms such as phosphorylation, acetylation,

ubiquitylation, or proteolysis of core components of the translation machinery(de Sousa

Abreu et al., 2009). And, more remarkable, TargetScan list two transcript variants of CCR2,

only one of which contains miR-125b target sites, the 3′UTR sequence we study belongs to

CCR2 (ENSG00000121807.5) which TargetScan considers as the less prevalent transcript,

therefore, considerations such as the percentage of the transcript variants to the function of

CCR2 and the mutual regulation of the two transcript variants still need further study.

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Importantly, further experiments should focus on the problems: Is there some 3'UTR variant

that can repress CCR2 translation, while stabilizing the 3'UTR allowing it to act as a ceRNA?

Is there cancer specific regulatory pathways that promote CCR2 protein degradation, while

stabilizing the mRNA?

As STARD13 is a major antagonist of RhoA-ROCK1 signaling, we examined whether

CCR2 can inactivate this pathway via acting as a STARD13 ceRNA. Intriguingly, CCR2

3′UTR inhibits RhoA activity rather than the expression of total RhoA and ROCK1. These

findings rule out the possibility of a direct action between CCR2 3′UTR and RhoA and

proved that CCR2 3′UTR-mediated inhibition of the RhoA activity is dependent on a

RhoGAP-containing protein instead of other mechanisms. RhoA and ROCK1 expression can

phosphorylate and inhibit myosin phosphatase (MYPT) and activate focal adhesion kinase

(FAK) in hypoxic breast cancer cells (Gilkes et al., 2014). Further research is needed to

confirm whether the phosphorylation of MYPT and FAK will be affected by CCR2 3′UTR.

CCR2 3′UTR-induced decrease of pMLCS19 and inhibition of stress fiber formation were

abolished by STARD13 siRNA, indicating that STARD13 acted as a downstream effector of

CCR2 3′UTR. Since our previous study has confirmed that a STARD13-correlated ceRNA

network can inhibit breast cancer metastasis (Li et al., 2016b), further investigation should be

carried out to confirm the regulation between CCR2 and STARD13-correlated ceRNA

network.

Although several clinical trials targeting CCL2-CCR2 signaling axis have been

established, CCL2-CCR2 signaling network is undeniably far more complex than that given

by the current researches (Lim et al., 2016). It still has a long way prior to set up CCL2 or

CCR2 targeted therapies successfully. The non-coding function of mRNA might create

functional complexity and diversification in both physiological and pathological conditions

and the unappreciated genetic dimension should be considered when designing treatment

strategy.

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4. Materials and Methods

4.1 Cell lines and culture

Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from ATCC

(Manassas, Virginia, USA) and maintained in high glucose DMEM and L15 medium (Gibco),

respectively. Both of the two mediums were supplemented with 10 % FBS (Gibco), 80 U/ml

penicillin and 0.08 mg/ml streptomycin and cells were incubated at 5 % CO2 at 37 °C. L15

medium with 0.6 µg/ml puromycin was used to incubate MDA-MB-231(C) cells (stably

transfected with CCR2 3′UTR), MDA-MB-231(V) cells (stably transfected with an empty

vector) and MDA-MB-231(S) (stably transfected with STARD13 3′UTR).

4.2 MicroRNA, siRNA, plasmids and transfection

MicroRNA mimics/ inhibitor, mimics NC/ inhibitor NC, siRNA and siRNA NC (NC)

synthesized in Biomics Biotechnology Inc., China. CCR2 siRNA (sense strand:

5′-CAUCAAUCCCAUCAUCUAU-3′), STARD13 siRNA (sense strand:

5′-CACCUUUCCAUCUCCUAAU-3′), DICER siRNA (sense strand:

5′-AAGGCUUACCUUCUCCAGGCU-3′). 3′UTR of CCR2 (NM_001123396.1) and

STARD13 3′UTR were cloned into the pSilencer 4.1 vector and the pCDNA 3.1 vector

respectively and the constructs were verified by DNA sequencing. The recombinant plasmids

were denoted CCR2 3′UTR and STARD13 3′UTR, respectively. CCR2 3′UTR with mutation

of all three predicted miR-125b binding sites were cloned into the pSilencer 4.1 vector and

was denoted CCR2 3′UTR MUT. Lipofectamine 2000 Reagent (Invitrogen) was used for the

microRNA mimics/ inhibitor, mimics NC/ inhibitor NC, siRNA, NC and plasmids

transfection. 48 h after transfection, the mRNA expression was detected by qRT-PCR, and the

protein expression was tested by Western blotting. 24 h after transfection, cells were treated

with TGF-β1 (PeproTech) at a concentration of 10 ng/ mL for 48 h.

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4.3 Adhesion assay

Wells of microwell plate were coated with matrix gel (BD Biosciences) 37 °C, 4 h, and

then were blocked for 1h with 1.2 % BSA in PBS. Cells transfected with CCR2 3′UTR or

pSilencer 4.1 were suspended at concentration of 0.6×105 cells/well in serum-free medium.

The colorimetric MTT-assay was used to determine the number of adherent cells after 1 h

adhesion.

4.4 Wound healing assay

Wound healing assay was conducted as described previously (Chou et al., 2016). MCF-7

and MDA-MB-231 cells were seeded in 6-well plates and transiently transfected with CCR2

3′UTR or pSilencer 4.1. Cells were allowed to grow to 90 % confluency in complete medium.

A vertical wound was made to cells with a sterile pipette tip and wounded monolayer was

washed with PBS to remove cell debris, and serum-free medium was used to maintained

cells, phase-contrast images were taken of each sampler at 0 h and 12 h (MDA-MB-231) or

24 h (MCF-7) at the same position of the wound. The distances that cells migrated into

wound surface were calculated for three different positions. Migration rate = (D0 - Dt) / D0, of

which, D0 stands for the distance measured at 0 h and Dt refers to the distance measured at 12

h (MDA-MB-231) and 24 h (MCF-7).

4.5 Transwell migration and invasion assay

The migration assay was performed using transwell insert chamber (a pore size of 8 µm;

Millipore) and the invasion assay was performed using BD Biosciences matrigel invasion

chambers (a pore size of 8 µm; BD Biosciences). After transfection, a total of 1×105 cells in

serum-free medium were placed into the upper chamber, the lower chamber was filled with

complete medium containing 20 % FBS. For migration assay, cells were allowed to migrate

at 37 °C for 24 h (MDA-MB-231) or 36 h (MCF-7). For invasion assay, cells were allowed to

invade at 37 °C for 36 h (MDA-MB-231) or 48 h (MCF-7). The migrated or invaded cells

were fixed in methanol for 30 min and stained with 0.1 % crystal violet for 30 min. The

stained cells were photographed, and the dye was eluted with glacial acetic acid and

quantified by measuring with Microplate Reader. (OD570 nm).

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4.6 Flow cytometric analysis of the cell cycle and apoptosis

Cells were harvested 48 h after transfection. The cell cycle and apoptosis were analyzed

by FACS caliber machine (Becton Dickinson) with cell cycle assay kit (Vazyme) and

apoptosis detection kit (Vazyme), respectively. The cell cycle distribution was determined

with propidium iodide (PI) staining. A total of 20,000 gated events were acquired to assess

the proportions of cells in different stages of the cell cycle and cell cycle profiles were

calculated by using the ModFit LT 4.0 software. For apoptosis assay, the cells were stained

with Annexin V-FITC and PI. 10,000 cells were collected and FlowJo software was used to

analyze the data and the data were expressed as cell percentage.

4.7 Luciferase reporter assay

To test whether the miRNA-125b bound directly to the 3′UTR of CCR2 mRNA, the

pMIR-REPORTTM miRNA Expression Reporter Vector System (Applied Biosystems)

consisting of an experimental firefly luciferase reporter vector and an associated β-gal

reporter control plasmid were used. Portion of the 3′UTR of CCR2 mRNA containing wild

type (WT) or mutant (MUT) miRNA-125b binding site was cloned respectively into the

firefly luciferase reporter vector. For the luciferase assay, MCF-7 or MDA-MB-231 cells in

24-well plates were cotransfected with recombinant firefly luciferase reporter plasmids

(CCR2-WT or CCR2-MUT), miRNA-125b mimics or mimics NC and β-gal reporter control

plasmid using lipofectamine 2000 reagent. Luciferase activity was measured according to the

manufacturer’s protocol and normalized to β-gal.

4.8 mRNA and miRNA quantification

Total RNA of cells after transfection was isolated by TRIzol reagent (Invitrogen). cDNA

was synthesized using the M-MLV (Promega) following standard protocols. Quantitative

PCR was performed by using Applied Biosystems StepOnePlusTM system. EzOmics SYBR

qPCR mix and miRNA-125b qPCR kit were purchased from Biomics. MicroRNA-125b

levels were normalized to U6 snRNA. The primers of various genes were designed and

synthesized as follows:

GAPDH: 5′-AAGGTCGGAGTCAACGGATT-3′, and 5′-CTGGAAGATGGTGATGGGAT

T-3′; CCR2: 5′-CTGTCCACATCTCGTTCTCGGTTTA-3′, and 5′-CCCAAAGACCCACT

CATTTGCAGC-3′; STARD13: 5′-AGCCCCTGCCTCAAAGTATT-3′, and 5′-ATGGGCG

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TCATCTGATTCTC-3′; RhoA: 5′-GGACCCCAGAAGTCAAGCAT-3′, and 5′-GAGCAG

CTCTCGTAGCCATT-3′; ROCK1: 5′-AACATGCTGCTGGATAAATCTGG-3′, and 5′-TG

TATCACATCGTACCATGCCT-3′; CCR2 3′UTR: 5′-ATGCCTCATTACCTTGT

G-3′, and 5′-CCATTCATCTGTGCCTGT-3′. STARD13 3′UTR: 5′-TTAAAGCTAGATGA

GGAGTTGCC -3′, and 5′-TTGACTTGGGGTCTGTAGTGATG-3′.

Gene relative expression level of each group was calculated using the 2-ΔΔct method.

4.9 Western blotting

Cells were harvested and lysed in buffer RIPA (Beyotime) on ice. Protein concentration

was measured using BCA method. 30 µg protein of each sample was fractionated by 10 % or

12 % SDS/PAGE and transferred electrophoretically onto polyvinylidene difluoride

membranes (Millipore). The membranes were blocked with 8 % BSA in Tris-buffered

saline/0.1 % tween 20 at 37 °C for 1h and then blotted with primary antibodies overnight at 4

°C. Antibodies against the following proteins were used: E-cadherin, N-cadherin and

vimentin (1:5000, Abcam); α-SMA (1:1000, Wanleibio); CCR2, MLC, pMLCS19 and RhoA

(1:1000, Cell Signaling Technology); β-actin (1:500, ZSGB-BIO); STARD13 (1:1000,

ABGENT). After incubation with horseradish peroxidase-conjugated secondary antibody

(ZSGB-BIO), the protein bands were detected using ECL chemiluminescence reagent

(Tanon). Protein expression levels were quantified by density analysis using Quantity One

Software and normalized to β-actin.

4.10 RNA-binding protein immunoprecipitation assay

RIP assay was performed using the Protein A/G Agarose Resin 4FF (YEASEN)

following the manufacturer’s protocol. Briefly, MDA-MB-231(C) cells (stably transfected

with CCR2 3′UTR), MDA-MB-231(V) cells (stably transfected with an empty vector) and

MDA-MB-231(S) (stably transfected with STARD13 3′UTR) cells were lysed by NP-40 lysis

buffer (Beyotime). Then, 100 µL cells extract was incubated with NP-40 buffer containing

Protein A/G Agarose Resin 4FF conjugated with human anti-Ago antibody (Cell Signaling

Technology) at 4 °C overnight. Agarose Resin were isolated by centrifugation and incubated

with proteinase K (Beyotime) to dissociate Ago2-RNA complex from the Agarose Resin.

qRT-PCR was performed to detect CCR2, STARD13 and miR-125b levels.

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4.11 RhoA GTPase assay

Cells were assayed for RhoA GTPase activity by RhoA G-LISA (Cytoskeleton)

according to the commercial protocol. Briefly, prepared lysates and measured lysate protein

concentration. Lysis buffer was added to equalize the cell extracts to give identical protein

concentrations in each sample. Samples were incubated in the wells provided and then

managed following the technical guide. Absorbance at 490 nm was measured using a

microplate spectrophotometer.

4.12 Immunofluorescence and F-actin visualization

Cells were fixed in 4 % paraformaldehyde for 20 min, permeabilized with 0.2 % Triton

X-100 for 20 min at 4 °C, and blocked with 3 % BSA in PBS for 1 h at room temperature.

For immunofluorescence, appropriate primary antibodies were used to blot overnight at 4 °C

and FITC-conjugated secondary antibody was used. For F-actin visualization, F-actin was

stained with Rhodamine phalloidin (Cytoskeleton) for 30 min at room temperature. The

nuclei were stained by DAPI for 30 min and observed with the confocal microscopy. Cells

were captured by adopting confocal laser scanning. Images were analyzed using OLYMPUS

FLUOVIEW ver.3.0 Viewer software and the fluorescent intensity was quantified using

Image J software.

4.13 In vivo metastasis study

All experiments were carried out under the ethical approval of Ethics Committee for

Animal Experimentation of China Pharmaceutical University. Six-week-old female nude

mice were purchased from Model Animal Research Center of Nanjing University.

Intravenous injection with MDA-MB-231 cells that were stably overexpressed CCR2 3′UTR

or empty vector was performed and 5×106 cells in 150 µl PBS were injected into 6 animals

per group. Eight weeks after injection, animals were scanned using the SkyScan 1176

micro-CT scanner. For bioluminescent analysis, Nano-GloTM substrate containing furimazine

(20 µg in 100 µl PBS, Promega) was injected intraperitoneally into mice before imaging.

Images of in vivo tumor growth and ex vivo lung metastasis were taken using the Carestream

noninvasive optical imaging system. Mice were sacrificed while lungs were sectioned and

stained with H&E.

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4.14 Statistical Analysis

The data were presented as the mean ± SD and statistical evaluation for data analysis

was determined by unpaired student’s test. P < 0.05 was considered to indicate a statistically

significant result. *P < 0.05, **P < 0.01, ***P < 0.001. ns indicate no significant differences

from control. Survival analysis was performed using the Kaplan-Meier plotter tool

(http://kmplot.com/analysis/).

5. Competing interests

The authors declare no competing or financial interests.

6. Funding

This work was supported by the National Major Special Program of New Drug Research

and Development (No. 2013ZX09301303-005), the Priority Academic Program Development

(PAPD) of Jiangsu Higher Education Institutions and Qing Lan Project, National Natural

Science Foundation of China, No. 81372331, and the Fundamental Research Funds for the

Central Universities, No. 2015ZD004.

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Fig. 1. Effects of CCR2 3′UTR on cells metastasis in MCF-7 and MDA-MB-231

cells. Good prognosis for breast cancer patients is associated with over expression of CCR2

(207794_at). Kaplan-Meier overall survival (A), distant metastasis free survival (B), relapse

free survival (C) probability for breast cancer patients with high (red) and low (black) CCR2

gene expression (divided at the median). The hazard ratio (HR) with 95 % confidence

intervals and logrank P were calculated for the low versus high CCR2 levels. (D) The cell

motility rate was measured by wound healing assay, movement of MCF-7 and MDA-MB-231

cells into the wound was shown for Blank (untransfected cells), CCR2 3′UTR (pSilencer 4.1

harboring CCR2 3′UTR sequence), pSilencer 4.1. (E) Quantification of the wound healing

assay for (D). (F) Effects of CCR2 3′UTR on adhesion to matrix gel of MCF-7 and

MDA-MB-231 cells. (G) Effects of CCR2 3′UTR on vertical migration and cell invasion in

MCF-7 and MDA-MB-231 cells were measured by transwell insert chamber and matrigel

invasion chambers respectively. (H and I) OD570 values of stain crystal violet for (G). Data

were presented as the mean ± SD, n =3,*p < 0.05, **p < 0.01, ***p < 0.001 vs. control

vector.

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Fig. 2. Ectopic expression of CCR2 3′UTR or STARD13 3′UTR hinders EMT of MCF-7

cells. (A) Western blot showing effects of CCR2 3′UTR on the expression of E-cadherin,

N-cadherin and α-SMA in MCF-7 cells (left panel); (right panel) Quantification of these

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proteins expression. (B) Western blot showing effects of STARD13 3′UTR on the expressions

of E-cadherin, N-cadherin and α-SMA in MCF-7 cells (left panel); (right panel)

Quantification of these proteins expression. (C) The expression of E-cadherin and N-cadherin

were detected by western blot in MCF-7 cells transfected with CCR2 3′UTR and then treated

with TGF-β1 for 48h (left panel); (right panel) Quantification of E-cadherin and N-cadherin

expression. (E) Immunofluorescence staining of E-cadherin and immunofluorescence

staining of filamentous actin (F-actin) with rhodamine-labeled phalloidin in MCF-7 cells

untreated or treated with TGF-β1 after being transfected with negative control pSilencer 4.1

or CCR2 3′UTR. Bars = 20 μm. Phase contrast images were shown. Data were presented as

the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control vectors.

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Fig. 3. Ectopic expression of CCR2 3′UTR or STARD13 3′UTR leads to the

alteration of EMT markers of MDA-MB-231 cells. (A) Western blot showing effects of

CCR2 3′UTR on the expressions of N-cadherin, Vimentin and α-SMA in MDA-MB-231 cells

(left panel); (right panel) Quantification of these proteins expression. (B) Western blot

showing effects of STARD13 3′UTR on the expressions of N-cadherin, Vimentin and α-SMA

in MDA-MB-231 cells (left panel); (right panel) Quantification of these proteins expression.

(C and D) Immunofluorescence staining of N-cadherin (C) and Vimentin (D) in

MDA-MB-231 cells after being transfected with negative control pSilencer 4.1 or CCR2

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3′UTR. Bars = 20 μm. Data were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01,

***p < 0.001 vs. control vector.

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Fig. 4. CCR2 3′UTR increases STARD13 expression and CCR2 knockdown decreases

STARD13 expression. (A and B) Overexpression of CCR2 3′UTR (A) or STARD13 3′UTR

(B) increased CCR2 and STARD13 mRNA levels in MDA-MB-231 cells, qRT-PCR analysis

for expressions of CCR2 and STARD13. (C and D) Overexpression of CCR2 3′UTR (left

panel of C) or STARD13 3′UTR (left panel of D) increased CCR2 and STARD13 protein

levels in MDA-MB-231 cells. Western analysis for CCR2 and STARD13 expressions and

β-actin as a loading control was shown; (right panel of C and D) Quantification of CCR2 and

STARD13 expressions. (E) STARD13 mRNA expression is efficiently reduced following

treatment of MDA-MB-231 cells with CCR2 siRNA, qRT-PCR analysis for expressions of

CCR2 and STARD13. (F) CCR2 knockdown decreased STARD13 protein levels in

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MDA-MB-231 cells (left panel of F). Western analysis for CCR2 and STARD13 expressions

and β-actin as a loading control was shown; (right panel of F) Quantification of CCR2 and

STARD13 expression. Data were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01,

***p < 0.001 vs. control vector.

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Fig. 5. miR-125b regulates CCR2 and STARD13 expression. (A) microRNA response

elements (MREs) shared by CCR2 and STARD13. 3′UTR of CCR2 and STARD13 have four

microRNA families in common and this table depicts the number of sites for each miRNA. (B

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and C) The transfection efficiency of miR-125b mimics (B) and miR-125b inhibitor (C) was

confirmed by qRT-PCR in MDA-MB-231 cells. Data were presented as the mean ± SD, n =3,

*p < 0.05, **p < 0.01, ***p < 0.001 vs. mimics NC or inhibitor NC. (D) Sketch of the

construction of CCR2-WT or CCR2-MUT vectors. Potential binding sites of miR-125b on

STARD13 3′UTR were shown. The binding sites were underlined. (E) After transfection,

luciferase reporter assay confirmed the direct binding between miR-125b and CCR2 in

MDA-MB-231 cells. Luciferase activity in cells was measured and normalized to β-gal

activity. Data were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs.

mimics NC. (F) RIP assay followed by microRNA qRT-PCR to detect the association of

miR-125b with CCR2 3′UTR or with STARD13 3′UTR. miR-17 was used as a negative

control since there is no binding site of miR-17 on CCR2 3′UTR or STARD13 3′UTR. Data

were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs.

MDA-MB-231(V). (G) miR-125b mimics reduced mRNA levels of CCR2 and STARD13 in

MDA-MB-231 cells. (H) miR-125b inhibitor increased mRNA levels of CCR2 and

STARD13 in MDA-MB-231 cells. (I and J) Western analysis of CCR2 and STARD13

expressions in MDA-MB-231 cells treated with miR-125b mimics (left panel of I) or

miR-125b inhibitor (left panel of J), β-actin expression was shown as a loading control; (right

panel of I and J) Quantification of these proteins expression. Data were presented as the mean

± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. mimics NC or inhibitor NC.

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Fig. 6. Regulation of STARD13 by CCR2 is microRNA dependent. (A and B) When

siCCR2 was co-transfected with siDICER, the mRNA (A) and protein (B) expressions of

STARD13 and CCR2 was examined by qRT-PCR analysis or western analyses. Data were

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presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC. (C and D)

Western analyses were used to detect CCR2 and STARD13 protein expressions in response to

CCR2 3′UTR (left panel of C) and STARD13 3′UTR (left panel of D) overexpression in

siDICER-treated MDA-MB-231 cells; (right panel of C and D) Quantification of protein

expression. (E) RIP assay followed by qRT-PCR to detect the enrichment of Ago2 on CCR2

3′UTR and STARD13 3′UTR in MDA-MB-231(V) cells and in MDA-MB-231(C) cells. (F)

Western analyses were performed to evaluate CCR2 and STARD13 protein expressions in

response to CCR2 3′UTR or CCR2 3′UTR MUT in MDA-MB-231cells (left panel of F);

(right panel of F) Quantification of protein expression. (G) The cell motility rate was

measured by wound healing assay, movement of MDA-MB-231 cells into the wound was

indicated for pSilencer 4.1, CCR2 3′UTR and CCR2 3′UTR MUT. Data were presented as the

mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control vector.

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Fig. 7. CCR2 3′UTR inhibits formation of F-actin stress fibers, RhoA activity and

MLC phosphorylation. (A) MDA-MB-231 cells were stained with rhodamine-labeled

phalloidin to detect F-actin stress fibers. Bars = 20 μm. (B and C) Western blot was used to

detect pMLCS19 protein in response to overexpression of CCR2 3′UTR (left panel of B) and

STARD13 3′UTR (left panel of C) in MDA-MB-231 cells; (right panel of B and C)

Quantification of pMLCS19 and MLC protein. (D and E) qRT-PCR was performed to quantify

RhoA and ROCK1 mRNA levels in MDA-MB-231 cells. (F and G) Western blot was

performed to examine total RhoA protein level in response to overexpression of CCR2

3′UTR (upper panel of F) and STARD13 3′UTR (upper panel of G) in MDA-MB-231 cells;

(lower panel of F and G) Quantification of RhoA protein. Data were presented as the mean ±

SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control vector. ns indicate no significant

differences from control vector (pSilencer 4.1 or pCDNA 3.1). (H) RhoA activation was

measured by G-LISA. Absorbance was read at 490 nm. Data were presented as the mean ±

SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control vector (pSilencer 4.1). (I)

MDA-MB-231 cells were transfected with siSTARD13 or NC in the presence or absence of

overexpression CCR2 3′UTR and stained with rhodamine-labeled phalloidin.

Immunofluorescence assay confirmed that siSTARD13 could reverse CCR2 3′UTR-induced

inhibition of F-actin. Bars = 20 μm. (J) siSTARD13 reversed CCR2 3′UTR –induced

down-regulation of phosphorylation of MLC protein in MDA-MB-231 cells (left panel of J);

(right panel of J) Quantification of pMLCS19 and MLC protein. Data were presented as the

mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC & pSilencer 4.1 or siSTARD13

& CCR2 3′UTR.

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Fig. 8. CCR2 3′UTR inhibits breast cancer metastasis in a xenograft model. (A)

qRT-PCR analysis for expression of CCR2 3′UTR in MDA-MB-231 cells stably transfected

with an empty vector or CCR2 3′UTR. Data were presented as mean ± SD, n =3, ***p <

0.001 vs. vector (B) Six mice were intravenously injected with MDA-MB-231 cells stably

transfected with an empty vector or CCR2 3′UTR. Eight weeks afterwards, animals were

scanned by micro-CT and representative transverse images were shown. The heart was

demarcated (‘H’) and the tumour were marked in red. (C) Representative bioluminescence

images of mice injected with MDA-MB-231(V) cells (stably transfected with an empty vector)

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or MDA-MB-231(C) cells (stably transfected with CCR2 3′UTR). Ventral views were shown.

(D) Ex vivo bioluminescent imaging showed the suppressive effect of CCR2 3′UTR

overexpression on pulmonary metastasis. (E) Representative histological images of lungs

from the series described in C. Enlarged image of representative fields (magnifications, ×5)

and representative fields (magnifications, ×20) were shown.

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Fig. S1. Flow cytometric detection of cell cycle and apoptosis of MCF-7 and MDA-MB-231

cells after transfection. Cells were transfected with CCR2 3′UTR (pSilencer 4.1 harboring CCR2

3′UTR sequence) or control vector pSilencer 4.1 for 48h. (A and C) Cells were processed for flow

cytometric analysis after PI staining and histograms showed cell distribution in G0/ G1, S and G2/

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M phases of the cell cycle. (B and D) DNA content quantif ication in G0/ G1, S and G2/ M phases

were shown. (E and G) Cells were stained with annexin V and PI. The numbers referred to the

percentage of cells in upper right ( later apoptosis) and lower right (early apoptosis) quadrants. (F)

The changes of the early, the later and the total apoptotic percentages in transfected MCF-7 cells.

(H) The changes of the early, the later and the total apoptotic percentages in transfected

MDA-MB-231 cells. Data were presented as the mean ± SD, n =3, ns indicate no signif icant

differences from control vector (pSilencer 4.1).

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Fig. S2. CCR2 3′UTR increases STARD13 expression and CCR2 knockdown decreases

STARD13 expression. (A and B) Overexpression of CCR2 3′UTR (A) or STARD13 3′UTR (B)

increased CCR2 and STARD13 mRNA levels in MCF-7 cells, qRT-PCR analysis for expressions

of CCR2 and STARD13. (C and D) Overexpression of CCR2 3′UTR (left panel of C) or

STARD13 3′UTR (left panel of D) increased CCR2 and STARD13 protein levels in MCF-7 cells.

Western analysis for CCR2 and STARD13 expressions and β-actin as a loading control was shown;

(right panel of C and D) Quantification of CCR2 and STARD13 expressions. (E) STARD13

mRNA expression is efficiently reduced following treatment of MCF-7 cells with CCR2 siRNA,

qRT-PCR analysis for expressions of CCR2 and STARD13. (F) CCR2 knockdown decreased

STARD13 protein levels in MCF-7 cells (left panel of F). Western analysis for CCR2 and

STARD13 expression and β-actin as a loading control was shown; (right panel of F)

Quantif ication of CCR2 and STARD13 expressions. Data were presented as the mean ± SD, n =3,

*p < 0.05, **p < 0.01, ***p < 0.001 vs. control vector.

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Fig. S3. miR-125b regulates CCR2 and STARD13 expressions. (A and B) The transfection

efficiency of miR-125b mimics (A) and miR-125b inhibitor (B) was confirmed by qRT-PCR in

MCF-7 cells. Data were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs.

mimics NC or inhibitor NC. (C) After transfection, luciferase reporter assay confirmed the direct

binding between miR-125b and CCR2 in MCF-7 cells. Luciferase activity in cells was measured

and normalized to β-gal activity. Data were presented as the mean ± SD, n =3, *p < 0.05, **p <

0.01, ***p < 0.001 vs. mimics NC or inhibitor NC. (D) miR-125b mimics reduced mRNA levels

of CCR2 and STARD13 in MCF-7 cells. (E) miR-125b inhibitor increased mRNA levels of CCR2

and STARD13 in MCF-7 cells. (F and G) Western analysis of CCR2 and STARD13 expressions in

MCF-7 cells treated with miR-125b mimics (upper panel of F) or miR-125b inhibitor (upper panel

of G), β-actin expression was shown as a loading control; (lower panel of F and G) Quantification

of these proteins expressions. Data were presented as the mean ± SD, n =3, *p < 0.05, **p < 0.01,

***p < 0.001 vs. mimics NC or inhibitor NC.

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Fig. S4. Regulation of STARD13 by CCR2 is microRNA dependent. (A and B) When siCCR2

was co-transfected with siDICER, the mRNA (A) and protein (B) expressions of CCR2 and

STARD13 were examined by qRT-PCR analysis. Data were presented as the mean ± SD, n =3, *p

< 0.05, **p < 0.01, ***p < 0.001 vs. NC. (C and D) Western analyses were used to detect CCR2

and STARD13 protein expression in response to CCR2 3′UTR (left panel of C) and STARD13

3′UTR (left panel of D) overexpression in siDICER-treated MCF-7 cells; (right panel of C and D)

Quantif ication of proteins expressions. Data were presented as the mean ± SD, n =3, *p < 0.05,

**p < 0.01, ***p < 0.001 vs. control vector.

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Fig. S5. CCR2 3′UTR inhibits formation of F-actin stress fibers, RhoA activity and MLC

phosphorylation. (A) MCF-7 cells were stained with rhodamine-labeled phalloidin to detect

F-actin stress fibers. Bars = 20 μm. (B and C) Western blot was used to detect pMLCS19

protein in

response to overexpression of CCR2 3′UTR (left panel of B) and STARD13 3′UTR (left panel of

C) in MCF-7 cells; (right panel of B and C) Quantification of pMLCS19

and MLC protein. (D and

E) qRT-PCR was performed to quantify RhoA and ROCK1 mRNA levels in MCF-7 cells. (F and

G) Western blot was performed to examine total RhoA protein level in response to overexpression

of CCR2 3′UTR (upper panel of F) and STARD13 3′UTR (upper panel of G) in MCF-7 cells;

(lower panel of F and G) Quantification of RhoA protein. Data were presented as the mean ± SD,

n =3, *p < 0.05, **p < 0.01, *** p< 0.001 vs. control vector. ns indicate no signif icant differences

from control vector (pSilencer 4.1 or pCDNA 3.1). (H) MCF-7 cells were transfected with

siSTARD13 or NC in the presence or absence of overexpression CCR2 3′UTR and stained with

rhodamine-labeled phalloidin. Immunofluorescence assay confirmed that siSTARD13 could

reverse CCR2 3′UTR-induced inhibition of F-actin. Bars = 20 μm. (I) siSTARD13 reversed CCR2

3′UTR –induced down-regulation of phosphorylation of MLC protein in MCF-7 cells (left panel

of I); (right panel of I) Quantification of pMLCS19

and MLC protein. Data were presented as the

mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC & pSilencer 4.1 or siSTARD13 &

CCR2 3′UTR.

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Fig. S6. CCR2 3′UTR increases STARD13 expression and inhibits MLC phosphorylation,

formation of stress fibers and migration of MDA-MB-231 cells. (A) Western blot showed

CCR2 and STARD13 protein in MDA-MB-231(V)

cells (stably transfected with an empty vector)

or MDA-MB-231(C)

cells (stably transfected with CCR2 3′UTR) (left panel of A); (right panel of A)

Quantif ication of CCR2 and STARD13 protein. (B) Western blot showed pMLCS19

and MLC

protein in MDA-MB-231(V)

cells (stably transfected with an empty vector) or MDA-MB-231(C)

cells (stably transfected with CCR2 3′UTR) (left panel of B); (right panel of B) Quantification of

pMLCS19

and MLC protein. (C) MDA-MB-231(V)

cells and MDA-MB-231(C)

cells were stained

with rhodamine-labeled phalloidin to detect F-actin stress fibers. Phase contrast images were

shown. Bars = 20 μm. (D) The cell motility rate was measured by wound healing assay,

movement of cells into the wound was shown for MDA-MB-231(V)

cells (stably transfected with

an empty vector), MDA-MB-231(C)

(stably transfected with CCR2 3′UTR). Data were presented

as the mean ± SD, n =3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. vector. ns indicate no signif icant

differences from vector.

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Fig. S7. Proposed model for how CCR2 3′UTR through RhoA-ROCK1 signaling regulates

motility of breast cancer cells. CCR2 3′UTR regulates STARD13 expression through in a

miR-125b-dependent manner. STARD13 inhibit RhoA-ROCK1 signaling activity through its

RhoGAP domain, leading to inhibiting the RhoA-mediated assembly of actin stress fibers and

MLC phosphorylation. The pathways converge to suppress cell motility, an essential step required

for cell migration and invasion and cancer metastasis. Rectangle box: ceRNA crosstalk of CCR2,

miRNA-125b and STARD13. Ovals box: RhoA-ROCK1 signaling pathway.

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